The mechanisms governing the spatial and temporal control of non-muscle myosin 2 (NM2) filament assembly in living cells are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we describe a sequential amplification mechanism for NM2 filament assembly within lamella wherein individual filaments emanating from single nucleation events continuously partition to form filament clusters that then populate large scale actomyosin structures deeper in the cell. Live, two-color imaging demonstrates that individual partitioning events coincide spatially and temporally with the movements of underlying actin fibers, and inhibition of actin dynamics suppresses partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibers to which they are bound. Both partition frequency and the rate of filament growth in the lamella are dependent on MLCK activity. Importantly, we provide direct evidence that the pool of NM2A monomer available for filament assembly is limiting, such that MLCK competes with Rho Kinase acting deeper in the cytoplasm for monomer to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and regulation of NM2 filament assembly in cells. Class 18A myosins (M18A) are a poorly understood class of myosin with domain architecture similar to that of myosin 2 (M2). Specifically, both M18A and M18A, two M18A isoforms generated by alternative splicing, consist of a motor domain followed by a short neck region and an extended coiled-coil domain that drives dimerization. Unlike M2, however, they possess a C-terminal non-helical tailpiece that harbors binding sites for SH3 and PDZ domain-containing proteins. Moreover, M18A also possesses an N-terminal extension containing a KE-rich region, an ATP-insensitive actin-binding site, and a PDZ domain. Knockout of M18A results in embryonic lethality in both mice and flies, suggesting a fundamental role in development. Despite their overall structural similarity to M2, M18A isoforms have no actin-activated ATPase activity and do not translocate actin filaments in vitro, suggesting that their functions do not require motor activity. Moreover, M18A isoforms do not assemble into filaments on their own. M18A isoforms do, however, co-assemble with M2 both in vitro and in vivo to form mixed bipolar filaments (Billington and Beach et al, Curr. Biol. 2015). This critical finding suggests that M18A isoforms may serve to regulate M2 filament turnover and/or act as adaptors to link M2 filaments to different cellular structures/signaling molecules via their extra N- and C-terminal domains, all without interfering with M2 motor activity. M18A is ubiquitously expressed across mammalian tissues, with elevated expression and isoform-specific expression in numerous cell types, including epithelia. In this study we determined the subcellular localization of M18A in polarized MDCK cell sheets and in cryo-sections of various mouse epithelia using an M18A-specific antibody. We find M18A concentrated at cell: cell junctions near the apical surface of polarized MDCK cells, a site where M2 is known to be critical for maintaining the integrity of adherens junctions. Using a CRISPR approach, we generated M18A null MDCKII lines and tested mature monolayers for barrier function. Both trans-epithelial resistance and FLUX were affected in M18A null cells. We also find that M18A is enriched in kidney proximal tubules and localizes with M2 on secretory granules in secretory tissues such as the pancreas and salivary gland. Finally, we find that M18A localizes along with M2 to cell: cell junctions in intestinal brush border epithelium. To investigate M18A function in the gut, we established a long-term intestinal enteroid culture system, a pertinent model to study epithelial cell proliferation, migration, and differentiation. Our focus now is on how M18A may be working together with M2 in two conserved processes in epithelial physiology and homeostasis: apical cell extrusion and interkinetic nuclear migration.